STANDARDIZATION OF AYURVEDIC DRUGS:

CHARACTERIZATION OF DASAMOOLARISTA

BY

TENNAKOON MUDIYANSELAGE SAMANTHA GOME TENNAKOON

M.Phil 2002

I DEDICATE THIS THESIS TO

MY PARENTS

DECLARATION

THE WORK DESCRIBED IN THIS THESIS WAS CARRIED OUT BY ME

UNDER THE SURPERVISION OF PROF. A.M. ABEYSEKERA AND PROF.

K.T.D. DE SILVA OF THE DEPARTMENT OF CHEMISTRY, UNIVERSITY OF

SRI JAYEWARDENEPURA AND A REPORT ON THIS HAS NOT BEEN

SUBMITTED TO ANY UNiVERSITY FOR ANOTHER DEGREE.

T.M NAKOON DATE: 31 l0\\0

DECLARATION

WE CERTIFY THAT THE ABOVE STATEMENT MADE BY THE CANDIDATE

IS TRUE AND THAT THIS THESIS IS SUITABLE FOR SUBMISSION TO THE

UNIVERSITY FOR THE PURPOSE OF EVALUATION.

dp" PROF. A.M. ABEYSE

DATE:

PROF. K.T.D. DE SILVA

DATE: 31(01/02

UI DV 2002

STANDARDIZATION OF AYURVEDIC DRUGS:

CHARACTERIZATION OF DASAMOOLARISTA

BY

TENNAKOON MUDIYANSELAGE SAMANTHA GOME TENNAKOON

Thesis submitted to the University of Sri Jayawardenapura

for the award of the degree of Master of Philosophy in Chemistry.

January 2002

CONTENTS PAGE

LIST OF CONTENTS

LIST OF TABLES ix

LIST OF FIGURES x

ACKNOWLEDGEMENTS xiv

ABBREVIATIONS xvi

ABSTRACT xvii

1. Introduction 1

1.1 Ayurveda and traditional medicine 1

1.2 Basic concepts of Ayurvedic medicine 1

1.2.1 Tridosha concept 2

1.2.2 Saptha dahtu concept 3

1.2.3 Mala concept 3

1.2.4 Srota concept 3

1.2.5 Agni concept 4

1.3 Pharmacology in Ayurveda 4

1.4 Ayurvedic pharmaceutical preparations 5

1.5 Ayurvedic Pharmacopoeias 6

1.5.1 Comparison between niganthus and modem pharmacopoeias 7

1.6 Traditional medicine(TM) in the world today 9

1.6.1 Barriers to Ayurvedic(AV) drugs 10

1.7 The status of Ayurveda in Sri Lanka 11

11

1.7.1 Health services and health problems in Sri Lanka 12

1.8 Cultivation of medicinal plants 14

1.9 Need for standardization 14

1.9.1 Problems and methods in the standardization of Ayurvedic drugs 18

1.10 Arista 23

1.10.1 Preparation of decoction 24

1.10.2 Fermentation 25

1.10.3 Sedimentation 26

1.11 Dasamoolarista(DMA) 26

2.0 Materials and Methods 28

2.1. Spectra 28

2.2 High performance liquid chromatography(HPLC) 28

2.3 Gas liquid chromatography (GLC) 28

2.4 Densitometry 28

2.5 Melting points 29

2.6 Thin layer chromatography(TLC) 29

2.6.1 Chromatographic reagents 29

2.7 Column chromatography 31

2.8 Plant materials and other ingredients 31

2.9 Dasamoolarista (DMA) samples 31

2.10 Solvents 32

2.11 Standard curves 32

111

2.12 Identity tests 32

2.12.1 Solvents(n-hexane, methylene chloride, choloroform, diethyl ether, 32

ethyl acetate) extract of DMA

2.12.2 Extract of the phenolic fraction of DMA 33

2.12.3 Aqueous extracts of plant ingredients of DMA 33

2.12.4 Laboratory scale preparation of the decoction of DMA 33

2.13 Isolation of marker and representative compounds 33

2.13.1 Kaempferol(i) and Quercetin(i)

33

2.13.2 Isoliquiritigenin(iii) 35

2.13.3 Plumbagin(iv) and a 4:1 mixture of Isoshinanolone(v) and 36

Epiisoshinanolone(vi) (ISMIX)

2.13.4 Acetylation of ISMIX 38

2.13.5 5-Hydroxrnethylfurfural(vii)(5-HMF) 39

2.13.6 Umbelliferone (viii) 40

2.13.7 Gallic acid (ix) 40

2.13.8 Isolation of dehydrocostus lactone(x) and 42

dihydrodehydrocostus lactone(xi)

2.13.9 Reduction of dehydrocostus lactone(x) to 43

dihydrodehydrocostus lactone(xi)

2.13.10 Alizarin(xii) and Purpurin (xiii) 44

2.14 Quantitative determination of marker and representative compounds 46

2.14.1 TLC-densitometric quantification 46

lv

2.14.1.1 Isoliquiritigenin(iii) by TLC-VIS method 47

2.14.1.2 Gallic acid (ix) by TLC-UV method 49

2.14.1.3 Gallic acid(ix) by TLC-FD method 51

2.14.1.4 5- Hydroxymethylfurfural(vii) (5-HMF) by TLC-UV method 53

2.14.1.5 Costuslactones by TLC-VIS method

55

2.14.1.6 Umbelliferone (viii) by Thin layer chromatography-Fluorescence 57

densitometery (TLC-FD) method

2.14.1.7 Mixture of Isoshinanolone(v) and epiisoshinanolone(vj) (ISMIX) 59

by TLC-FD method

2.14.2 HPLC quantification 61

2.14.2.1 Gallic acid(ix) in DMA and its decoction 62

2.14.2.2 5-HMF(viii) in DMA and its decoction 64

2.14.2.3 Preparation of Pluinbago indica L. root extract for determining 66

the ratio of isoshinanolone(v) and epiisoshinanolone(vi)

2.14.3 GLC Quantification 67

2.14.3.1 Dehydrocostus lactone(x)(DHC) and dihydrodehydrocostus lactone(xi) 68

(DDHC) by GLC

2.14.3.2 Monitoring of DHC and DDHC contents during industrial 71

scale manufacture of DMA (at Link Natural Products)

2.14.3.2.1 Determination of DHC content in decoction 71

2.14.3.2.2 Determination of DHC content in fermenting decoction 71

LVA

2.14.3.2.3 Determination of DHC content in fermented decoction 72

(DMA) samples preparation for GLC analysis

2.14.3.2.4 Preparation of a mixture of DHC and DDHC extract from 72

Saussurea lappa C.B. Clarke.rhizomes

2.15 Preparation of extracts for chromatographic analysis of plant 72

with problems of identity

2.15.1 Methanol extract of Acacia catechu WilId, 72

Myroxylon balsam um(L.)Harms heartwoods

2.15.2 Hydrodistillation of Nardostachys jatamansi DC .rhizome 73

2.15.3 Chloroform extract of Saussurea lappa C.B. Clarke 73

and Inula racemosa Hook.f.rhizomes

2.15.4 Chloroform extract of Nardostachysjatamansi DC and 73

Flickingeria macraei(Lindely) Seidenf.rhizomes

2.15.5 Methanol extract of Hem idesmus indicus R.Br. and 74

Crytolepis buchanani Rome& Shults. roots

2.15.6. Diethyl ether extract of bees honey 74

2.16 Development of Fingerprints (FP)

75

2.16.1 GLC fingerprints(GLC-FP)

75

2.16.2 HPLC fingerprints(HPLC-FP)

75

2.16.3 TLC fingerprints(TLC-FP)

75

vi

3.0 Results and Discussion

3.1 Development of Identity tests

3.1.1 Identity tests based on TLC

3.1.1.1 Identity tests for Plum bago indica L.

3.1.1.2 Identity tests for Aegle marmelos Correa.

3.1.1.3 Identity tests for Saussurea lappa C.B. Clarke.

3.1.1.4 Identity tests for Woodfordiafruticosa (L.)Kurz.

3.1.1.5. Identity test for Pterocarpus marsupium Roxb.

3.1.1.6 Identity tests for Rubia cordifolia L.

3.1.1.7 Identity tests for Woodfordiafruticosa (Linn) Kurz. and

Glycyrrihza glabra L.

3.1.1.8 Identity test for Eugenia caryophyllata Thumb. and

Cinnamornuin verurn Presl.

3.1.1.9 Identity tests for Pterocarpus inarsupium Roxb.,

Glycyrrihza glabra L. and Myroxylon balsam urn (L.) Harms.

3.1 .2 Identity tests based on GLC

3.1.2.1 Tests for compounds other than marker and compounds

in DMA

3.1.2.1.1 Gallic acid (ix)

3.1.2.1.2 5-Hydroxymethyl furfural(5-HMF) (vii)

3.2 Quantitative analysis

3.2.1 Quantitative determination of gallic acid(ix)

76

76

77

77

79

81

82

83

84

85

I

vii

3.2.2 Quantitative determination of 5-1-IMF (vii) 92

3.2.3 Quantitative determination of Isoshinanolone(v) and 94

Epiisoshinanolone(vi)

3.2.4 Quantitative determination of dehydrocostus lactone(x) and 101

dihydrodehydrocostus lactone(xi)

3.2.5 Quantitative determination of umbelliferone(viii) 108

3.2.6 Quantitative determination of isoliquiritigenin(iii) 110

3.3 Parameters for process control 112

3.3.1 Boiling process 112

3.4. Adulterants and substitutents 117

3.4..1 Nardostachysjatamansi D.0 and 119

Flickingeria macraei (Lindley) Seidenf.

3.4.2 Hemidesmus indicus R.Br. and 120

Ciyptolepis bun chanaii Roem. & Schult.

3.4.3 Bees honey 120

3.5 Chromatographic fingerprints for Dasamoolarista 121

3.6 Conclusion 135

3.7 Draft specifications for Dasamoolarista 137

References 138

Appendix-i Thin layer chromatograms 160

vii'

Appendix-ii 185

1H Nuclear magnetic resonance spectra('H NMR)

and 13 C Nuclear magnetic resonance spectra (13 C NIMR) for marker,

representative compounds and compounds other than makers or representatives

of Dasamoolarista

Appendix-ui List of plants used in the preparation of Arista 217

Appendix-iv Structural formulae of markers ,representative compounds 222

and compounds other than markers and representatives

lx

LIST OF TABLES

PAGE Table-I TLC densitometer settings 46

Table-2 HPLC settings 61

Table-3 GLC settings 67

Table-4 Gallic acid content of reference samples and commercial samples of 91

DMA determined by TLC-UV,TLC-FD and HPLC methods

Table -5 5-HMF content of reference samples and commercial samples of 93

DMA determined by TLC-UV and HPLC methods

Table -6 ISMIX content of reference samples and commercial samples of 97

DMA determined by TLC-FD method

Table - 7 ISMIX content of Plumbago indica L.root from two locations. 99

Table - 8 Ratio of isoshinanolone and epiisoshinanolone in Plumbago indica L. 100

root determined by HPLC and 1 H NMR

Table -9 Costuslactone content of reference samples and commercial 105

samples of DMA determined by GLC and TLC-VIS methods

Table- 10 Umbelliferone content of reference samples and commercial samples 109

of DMA determined by TLC-FD method

Table -11 Isoliquiritigenin content of reference samples and commercial samples 111

of DMA determined by TLC-VIS method

Table- 12 Official subsitutents, unofficial subsitutents and adulterants for 118

some crude drugs used in DMA

x

LIST OF FIGURES PAGE

Fig-I TLC-VIS densitometric standard curve for isoliquiritigenin 48

Fig-2 TLC-UV densitometric standard curve for gallic acid 50

Fig-3 TLC-FD densitometric standard curve for gallic acid 52

Fig-4 TLC-LTV densitometric standard curve for 5-HMF 54

Fig-5 TLC-VIS densitometric standard curve for dehydrocostus lactone 56

Fig-6 TLC-FD densitometric standard curve for umbelliferone 58

Fig-7 TLC-FD densitometric standard curve for isoshinanolone 60

Fig- 8 HPLC standard curve for gallic acid 63

Fig- 9 HPLC standard curve for 5-HMF 65

Fig- 10 GLC standard curve for dehydrocostus lactone 69

Fig- 11 GLC standard curve for dihydrodehydrocostus lactone 70

Fig-12 HPLC chromatogram and LC-UV spectra of mixture 95

of isoshinanolone and epi i soshinano lone

Fig -13 Stability of fluorescent intensity of ISMIX after treatment with ethanolic 96

aluminium hydroxide and ethanolic paraffin reagent

Fig -14 TLC-FD densitometric fingerprint for methanol extract of 100

Plumbago indica L .root

Fig-IS Stability of colour complex of mixture of DHC and DDHC with 101

LB spray reagent on TLC

Fig -16 TLC —densitogram of chloroform extract of reference DMA 103

- solvent system : hexane :diethylamine (80:20)

xi

Fig- 17 TLC —densitogram of chloroform extract of reference DMA 103

- solvent system : hexane :diethylamine (99.5: 0.5)

Fig —18 Visible spectra of dehydrocostus lactone and 104

dihydrodehydrocostus lactone after treatment of

Liebermarm Burchard reagent

Fig - 19 GLC chromatogram of methylene chloride extract of 107

Saussurea lappa C.B. Clarke rhizome.

Fig- 20 HPLC chromatogram(direct injection) for gallic acid and 5-HMF 113

in reference DMA

Fig -21 Variation of pH value and temperature in decoction of reference DMA 114

during boiling (R1 , R2, R3 are selected vessels)

Fig -22 Variation of gallic acid content of decoction of reference DMA 114

during boiling

Fig -23 Variation of 5-HMF content of decoction of reference DMA 114

during boiling

Fig- 24 Variation of dehydrocostus lactone content of decoction of reference 115

DMA during boiling

Fig- 25 GLC profile of methylene chloride extract of decoction of 116

reference DMA after 12 hours of boiling

Fig-26 Variation of dehydrocostus lactone content in fermenting decoction 116

xli

Fig -27 GLC profile of methylene chloride extract of decoction of reference 117

DMA after 10 days of the fermentation

Fig-28 GLC fingerprint of oil of Nardostachysjatamansi DC. 119

Fig-29 Fingerprint-I 126

Fig -30 Fingerprint -2 127

Fig- 31 Fingerprint - 3 127

Fig- 32Fingerprint - 4 128

Fig- 33 Fingerprint - 5 129

Fig-34 Fingerprint - 6 130

Fig -35 Fingerprint - 7 131

Fig-36 Fingerprint - 8 133

Fig-37 'H NMR spectrum of Kaempferol 186

Fig-38 13 C NMR spectrum of Kaempferol 187

Fig-39 'H NMR spectrum of Quercetin 188

Fig-40 13 C NMR spectrum of Quercetin 189

Fig-41 'H NMR spectrum of isoliquiritigenin 190

Fig-42 13 C NMR spectrum of isoliquiritigenin 191

Fig-43 'H NMR spectrum of Plumbagin 192

Fig-44 13 C NMR spectrum of Plumbagin 193

Fig-45 'H NMR spectrum of mixture of isoshinanolone and epiiso shinano lone 194

Fig-46 13 C NMR spectrum of mixture of isoshinanolone and epiisoshinanolone 195

Fig-47 'H NMR spectrum of isoshinanolone 196

Fig-48 13C NMR spectrum of isoshinanolone 197

Fig-491 H NMR spectrum of 5-HMF 198

Fig-50 13 C NMR spectrum of 5-HMF 199

Fig-5 1 'H NMR spectrum of Umbelliferone 200

Fig-52 13 C NMR spectrum of Umbelliferone 201

Fig-53 'H NMR spectrum of Gallic acid 202

Fig-54 13 C NMR spectrum of Gallic acid 203

Fig-55 'H NMR spectrum of dehydrocostus lactone 204

Fig-56 13C NMR spectrum of dehydrocostus lactone 205

Fig-57 'H NMR spectrum of dihydrodehydrocostus lactone 206

Fig-58 13C NMR spectrum of dihydrodehydrocostus lactone 207

Fig-59 DEPT 450 spectrum of dihydrodehydrocostus lactone 208

Fig-60 DEPT 900 spectrum of dihydrodehydrocostus lactone 209

Fig-61 DEPT 135° spectrum of dihydrodehydrocostus lactone 210

Fig-62 HETROCOSY spectrum of dihydrodehydrocostus lactone 211

Fig-63 HOMOCOSY spectrum of dihydrodehydrocostus lactone 212

Fig-64 'H NMR spectrum of the mixture of dehydrocostus lactone 213

and dihydrodehydrocostus lactone

Fig-65 13 C NMR spectrum of the mixture of dehydrocostus lactone 214

and dihydrodehydrocostus lactone

Fig-66 1 H NMR spectrum of Alizarin 215

Fig-67 13 C NMR spectrum of Alizarin 216

ACKNOWLEDGEMENTS

I wish to express my most sincere gratitude to Prof. A.M. Abeysekera and Prof. Tuley

De Silva, my supervisors, for their guidance and help during my work for this

dissertation. Their keenness and the encouragement extended to me with patience,

inspired me to proceed with the work to a successful completion, and their advice

given through out the project was extremely valuable.

I also wish to express my sincere gratitude to Dr. Devapriya Nugawela, Managing

Director, Link Natural Products (Pvt) Ltd, who gave me the opportunity, all facilities and

encouragement and co-funded this project along with the University of Sri

Jayewardenapure.

I thank the chemistry department of the University of Sri Jayewardenapure for providing

me with facilities to carry out my research. The cooperation of the academic and non-

academic staff of the chemistry department during my stay there is gratefully

acknowledged. Special mention must be made of Mr. Sri Lal Rangoda ,who maintained

the research laboratory. I also thank Mr. Janaka Nikawela and Mr. Janaka Abeysinghe

who were my research colleagues, for their support.

I wish to thank the Heads , Departments of Chemistry at the , Universities of Colombo

and Peradeniya, who extended the facilities of using their analytical instruments. My

sincere thanks are also due to Dr. Mrs. Thusita Wijeratne for her valuable assistance in

the interpretation of NMR spectra, and to Dr. Dammika Dissanayke who provided me

with valuable advice on the analytical techniques of gas chromatography.

xv

I am veiy greatful Mr. Wimal Pathmasiri who performed my NMR and GC/MS

experiments and provided me with literature from the Uppsala University library. His

assistance was invaluable throughout my project. I also would like to thank Mr. Jagath

Weerasena for sharing with me his valuable experiences in the field of chromatography.

I wish to thank the Curator ,National Herbarium Peradeniya and Prof. S.S. Handa ,RRL,

Jammu, India and Dr. A. K. S .Rawat National Botanical Research Institute, Lucknow,

India for allowing me to use their herbaria.

I would like to acknowledge the help of the staff of Link Natural Products (Pvt) Ltd

who assisted me during the study and I specially thank Miss Yamuna Dasanayake for her

kind cooperation during the study of the manufacturing process.

I also wish to thank miss Elani Jayawardena and Miss P.W.N.W. Perera for all their

assistance.

I gratefully acknowledge the assistance rendered by Mr. W. Gamagae and Mr.

Mallikarachi at thesis preparation.

The assistance given by Mr.K.S. Wijenayake and Mr. Prabath in the documentation of

thin layer chromatograms is gratefully acknowledged.

I thank my brother Janaranjana and sisters-in-law, , Dilrukshi,Denesha and Deepika for

typing my thesis, and their assistance in other ways.

Finally, my deepest thanks goes to my wife Mayurani , for her patience in enduring my

many evenings away from home and her ever present encouragement.

xvi ABBREVIATIONS

5-HMF 5-Hydroxymethylfurfural AA Arjunarista ABA Abeyarista AKA Asokarista AMA Amurtarista ASA Asvagandarista AT-225 50% Cyanopropylmethyl,50%phenylmethylpolysiloxane AV Ayurvedic BA Balarista CDD Cosmetic Device and Drug Act DA Danthyarista DDHC Dihydrodehydrocostus lactone DDMIX A mixture of dehydrocostus lactone and

dihydrodehydrocostus lactone DHC Dehydrocostus lactone DMA Dasamoolarista DRA Draksharista PD Fluorescence densitometry FDC Food Drug and Cosmetic Act PP Fingerprint GLC-FP Gas Liquid Chromatography- Fingerprint HPLC-FP High performance chromatography- Fingerprint IS Internal Standard ISMIX A mixture of isoshinanolone and epiisoshinanolone KA Kadirarista LB Liebermann Burchard reagent MA Musthkarista NA Not applicable NBA Nimbarista ND Not dectected NP/PEG Natural products/polyethyleneglycol reagent perp. TLC Preparative Thin Layer Chromatography Rt Retention time SA Saraswatharista sh Shift TC-1 100 % dimethylpolysiloxane TLC-FD Thin Layer chromatography-Fluorescence densitometry TLC-FP Thin Layer chromatography-Fingerprint TLC-UV Thin Layer chromatography-Ultraviolet spectroscopy TLC-VIS Thin Layer chromatography- Visible spectroscopy

xvii

STANDARDIZATION OF AYURVEDIC DRUGS:

CHARACTERIZATION OF DASAMOOLARISTA

TENNAKOON MUDJYANSELAGE SAMANTHA GOME TENNAKOON

ABSTRACT

Ayurveda plays a significant role in the health care system in Sri Lanka. Within the social

context in which Ayurveda is practised in the present day, the standardization and quality

assurance of Ayurvedic drugs is urgent and imperative.

Dasamoolarista (DMA) ) is a complex drug containing over 60 ingredients. Tests for

identity in complex herbal drugs such as DMA can be devised through chromatographic

methods to identify specific marker compounds which can be correlated with specific

plant ingredients and representative compounds for groups of plant ingredients. A

strategy for identifying such marker compounds by comparing the thin layer

chromatograms of drugs(Arista) having closely related formulae, was developed. Thin

layer chromatographic systems to detected the following plant ingredients through the

marker compounds and representative compounds shown in parenthesis were developed;

Aegle marmelos Correa. (Umbelliferone); Plumbago indica L.(Isoshinanolone and

Epiisohinanolone); Saussurea lappa C.B. Clarke. (Dehydrocostus lactone and

Dihydrodehydrocostus lactone); Glycyrrhiza glabra L. , Plerocarpus rnarsupiuln Roxb.,

and Myroxyluin balsamum (L.) Harms. (Isoliquiritigenin); Rubia cordfo1ia L. (Alizarin

and Purpurin); Eugenia caryophyllata Thumb, and Cinnamonium verurn Presl (Eugenol);

xviii

Woodfordiafruticosa (Linn.)Kurz., Vitis vinfera L. and bees honey ( Quercetin and

Kaernpferol).

It is proposed that tests for strength(potency) of drugs such as DMA of unknown

pharmacological action, can consist of quantitative measurements of compounds of high

biological activity found in the drug and specific marker compounds irrespective of their

biological activity. Analytical methods based on TLC densitometry ,HPLC and GLC

were used to quantify gallic acid, isoliquiritigenin, umbelliferone, ,dehydrocostus lactone,

dihydrodehydrocostus lactone, 5-hydroxymethylfurfural , isoshinanolone and

epiisoshinanolone in DMA. The analytical methods were shown to be precise and

accurate. These methods were then used to study the variability in composition of

different commercial brands of DMA, and of different manufacturing batches of DMA of

the same brand. Inter batch and inter brand variability was high, indicating a significant

variation in the quality of crude drugs used in the manufacture of the drug. Changes in

the level of dehydrocostus lactone, gallic acid and 5-hydroxymethylfurfural during the

different stages of manufacture were monitored. It was concluded that levels of gallic

acid and 5-hydroxymethylfurfural were more suitable as parameters for process control

than those of dehydrocostus lactone.

Finally, eight chromatographic fingerprints covering a wide range of compounds were

developed which could be used for routine quality control, and would provide an overall

measure of identity and potency.